With a significant increase in the average life span during last century, more and more people are facing old age and diseases associated with aging. By elucidating the mechanisms of aging, it may eventually become possible to delay the deleterious effects from old age through rational drug intervention. Because the phenotype of mice deficient in Klotho protein suggests that Klotho suppresses aging, identification of the Klotho protein function may provide new insights into the mechanisms behind aging required to achieve this long-term goal. Klotho-deficient mice exhibit a syndrome resembling human aging such as a shortened life span, arteriosclerosis, osteoporosis, skin atrophy, ectopic calcification, lipodystrophy, and pulmonary emphysema. Consequently, the klotho mouse becomes the first laboratory animal model of human aging caused by a single gene mutation. Recent evidence suggests that Klotho protein functions as a humoral factor that inhibits insulin signaling and reduces oxidative stress. Interestingly, these two effects of Klotho are identical with the two evolutionally conserved mechanisms that can suppress aging in lower animals. The proposed mechanism of Klotho action is that Klotho binds to its putative cell-surface receptor and sends a signal into the cell to inhibit insulin signaling and suppress oxidative stress, which eventually suppresses aging in mammals. The objective of this proposal is to test this hypothesis. Specific aims are to: (1) Determine the mechanism by which Klotho inhibits insulin action. Phenotypic consequence of Klotho-deficient mice whose insulin-signaling pathway is chronically inhibited is also determined. If inhibition of insulin signaling is essential to the anti-aging effect of Klotho, the aging phenotypes would be improved in these mice. (2) Determine the mechanism by which Klotho reduces oxidative stress. Phenotypic consequence of Klotho-deficient mice overexpressing an enzyme that detoxifies reactive oxygen species is also determined. If reduction in oxidative stress is essential to the anti-aging effect of Klotho, the aging phenotypes would be improved in these mice. These studies would provide the first genetic evidence for the involvement of insulin signaling and oxidative stress in mammalian aging. (3) Identify Klotho receptor. Identification of Klotho receptor is indispensable to elucidating intracellular Klotho-signaling pathway. In addition, it promises to promote better understanding of the Klotho protein function and verify its role as an anti-aging hormone, which would have a significant impact on basic research and medical practice of aging.